An important parameter in monitoring metabolic control in diabetes is a measurement of blood or serum fructosamine. Fructosamine is formed by non-enzymatic glycosylation of proteins by glucose. In addition, since the fructosamine linkages formed by this glycosylation are stable, fructosamine content is much less variable than glucose content. Therefore, fructosamine content is a useful index of the average glucose levels. An accurate means of fructosamine determination as provided by this invention is important in diagnosing and monitoring diabetes.
European Patent Publication No. 85 263 describes a simple method of determining the fructosamine content in serum samples. This method is based on the fact that fructosamines in alkaline solution form eneaminols which act as reducing agents on tetrazolium salts, such as for instance nitro-blue tetrazolium (NBT), thereby causing a change in color which can be photometrically determined, the measured change in color in a defined time interval being proportional to the quantity of fructosamines present in the sample.
This method however has a number of drawbacks. Problems may arise in particular when using serum as sample material, because other serum components, such as bilirubin, uric acid or proteins with free thiol groups also act as reducing agents on the color indicators or reagents. Besides, lipids, when present in high concentration, may cause turbidity of the sample, which has negative effects on photometric measurements and makes determination of the fructosamine content difficult, if not impossible.
To overcome these drawbacks, it has been proposed in European Patent Publication No. 309 882 to treat the sample before the color reaction, at a nearly neutral pH, with reagents which remove the interfering sample components, and then to set a basic pH which is necessary for the color reaction, and start the color reaction by adding the color indicator or reagent. This process is practically carried out with a set of two separate ready to use liquid reagents, the first reagent being an almost neutral buffer, which contains the reagents for removing the interfering sample components, and the second reagent being an alkaline buffer, which contains the color reagent. In order to use this process on automated clinical analysers, the color reagent and the alkaline buffer must be present in a relatively high concentration. It has been found however that the color reagents, usually tetrazolium salts, are not stable in alkaline medium in the presence of relatively high concentrations of color reagent and buffer salts, so that said second reagent is not shelf-stable.
In commercially available kits (Fructosamine Plus and Unimate Fructosamine, Hoffmann-La-Roche, Basel, Switzerland), this disadvantage has been avoided by separating the reagent components as follows: the first reagent consists of a tablet, which contains the color indicator and agents for removing non specifically reducing sample components, such as uricase; the second reagent component consists of an alkaline buffer which contains the remaining agents. In order to make the ready to use solution necessary for the analysis, the tablet (first reagent) must then first be dissolved in the second reagent. This way of proceeding gives excellent results but has following drawbacks: dissolution of the tablet takes at least 15 minutes and cannot be automated; the thus obtained solution of mixed reagents has a usable life time of less than two weeks.
There is therefore a need for reagents for determining the fructosamine content which do not have the above drawbacks. The problem to be solved by the present invention is to provide such reagents. This problem is solved by providing the set of reagents of this invention.